U.S. patent number 5,773,257 [Application Number 08/469,564] was granted by the patent office on 1998-06-30 for method for producing primed nucleic acid templates.
This patent grant is currently assigned to Stratagene. Invention is credited to Eric J. Mathur, Kirk B. Nielson.
United States Patent |
5,773,257 |
Nielson , et al. |
June 30, 1998 |
Method for producing primed nucleic acid templates
Abstract
The present invention relates to an improved method for
producing primed nucleic acid templates. Specifically, it relates
to a method, compositions and kits therefor, of increasing the
specificity of primer extension reactions by hybridizing primer to
template in the presence of single-stranded nucleic acid binding
protein.
Inventors: |
Nielson; Kirk B. (San Diego,
CA), Mathur; Eric J. (Solana Beach, CA) |
Assignee: |
Stratagene (La Jolla,
CA)
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Family
ID: |
23688364 |
Appl.
No.: |
08/469,564 |
Filed: |
June 6, 1995 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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224981 |
Apr 7, 1994 |
5646019 |
|
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425867 |
Oct 24, 1989 |
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Current U.S.
Class: |
435/91.1;
435/91.2; 530/350 |
Current CPC
Class: |
C12Q
1/6832 (20130101); C12Q 1/6848 (20130101); C12Q
1/686 (20130101); C12Q 1/686 (20130101); C12Q
1/6832 (20130101); C12Q 2527/125 (20130101); C12Q
2522/101 (20130101); C12Q 2563/137 (20130101); C12Q
2527/137 (20130101); C12Q 2522/101 (20130101) |
Current International
Class: |
C12Q
1/68 (20060101); C12P 019/34 (); C07K
014/195 () |
Field of
Search: |
;435/6,91.1,91.2
;530/350 ;935/77,78 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Beck, B.N. and Ho, S.N., "Increased specificity of PCR-amplified
products by size-fractionation of restriction enzyme-digested
template genomic DNA," Nucleic Acids Research 16(18):9051 (1988).
.
Haqqi, T.M. et al., "Specific amplification with PCR of a
refractory segment of genomic DNA," Nucleic Acids Research
16(24):11844 (1988). .
Chase, J.W. and Williams, K.R., "Single-stranded DNA binding
proteins required for DNA replication," Annu. Rev. Biochem
55:103-36 (1986). .
Chase, J. W. et al., "Characterization of the Escherichia coli
SSB-113 mutant single-stranded DNA-binding protein. Cloning of the
gene, DNA and protein sequence analysis, high pressure liquid
chromatography peptide mapping, and DNA-binding studies," Journal
of Biological Chemistry 259(2):805-14 (1984). .
Coleman, J.E. and Oakley, J.L., "Physical chemical studies of the
structure and function of DNA binding (helix-destabilizing)
proteins," CRC Crit. Rev. Biochem. 7(3):247-89 (1980). .
LaDuca, R.J. et al., "Site-specific pausing of deoxyribonucleic
acid synthesis catalyzed by four forms of Escherichia coli DNA
polymerase III," Biochemistry 22(22):5177-88 (1983). .
Muniyappa, K. et al., "Mechanism of the concerted action of recA
protein and helix-destabilizing proteins in homologous
recombination," Proc. Natl. Acad. of Sci. USA 81(9):2757-61 (1984).
.
Huberman J.A. et al., "Stimulation of T4 bacteriophage DNA
polymerase by the protein product of T4 gene 32," J. Mol. Biol.
62(1):39-52 (1971). .
Kaspar, P. et al., "An improved double stranded DNA sequencing
method using gene 32 protein," Nucleic Acids Research 17(9): 3616
(1989). .
Studier, F.W., "A strategy for high-volume sequencing of cosmid
DNAs: random and directed priming with a library of
oligonucleotides," Proc. Natl. Acad. Sci. USA 86(18):6917-21
(1989). .
Sommer, R. and Tautz, D., "Minimal homology requirements for PCR
primers," Nucleic Acids Research 17(16):6749 (1989). .
Christiansen, C. and Baldwin, R.L., "Catalysis of DNA reassociation
by the Escherichia coli DNA binding protein: A polyamine-dependent
reaction," J. Mol. Biol. 115(3):441-454 (1977). .
Lindberg, G. et al., "Purification and characterization of the
coliphage N4-coded single-stranded DNA binding protein," J. Biol.
Chem. 264(21): 12700-8 (1989). .
Hong, G.F., "Sequencing of large double-stranded DNA using the
dideoxy sequencing technique," Biosci Rep. 2(11):907-912 (1982).
.
Muta Gene M13 in vitro mutagenesis kit instructions version #189
89-0096, p. 34 Bio-Rad Laboratories, Richmond, CA. .
Innis, M.A., et al., "DNA Sequencing with Thermus aquaticus DNA
polymerase and direct sequencing of polymerase cahin
reaction-amplified DNA",PNAS 85:9436-9440, Dec. 1988..
|
Primary Examiner: Zitomer; Stephanie W.
Attorney, Agent or Firm: Halluin; Albert P. Pennie &
Edmonds LLP
Parent Case Text
This is a division of application Ser. No. 08/224,981, filed Apr.
7, 1994 now U.S. Pat. No. 5,646,019; which is a continuation
application of Ser. No. 07/425,867, filed Oct. 24, 1989, now
abandoned.
Claims
What is claimed is:
1. A method of preparing a strand of nucleic acid having a
nucleotide sequence substantially complementary to a template
nucleic acid, which method comprises:
(a) forming a primer extension reaction admixture whereby
nonspecific hybridization of polynucleotide synthesis primer is
reduced by admixing said template nucleic acid with a hybridization
effective amount of (i) said polynucleotide synthesis primer having
a nucleotide sequence complementary to a portion of the template
nucleic acid, (ii) an isolated single-stranded nucleic acid binding
protein (SSB), (iii) a divalent cation, and (iv) an enzyme capable
of inducing polynucleotide synthesis in the presence of a primed
template; and
(b) maintaining the primer extension reaction admixture under
polynucleotide synthesizing conditions for a time period sufficient
for said enzyme to produce a primer extension product, thereby
producing said strand of nucleic acid.
2. The method of claim 1 wherein said single-stranded nucleic acid
binding protein is Eco SSB.
3. The method of claim 1 wherein said divalent cation is selected
from the group consisting of Mg.sup.++, Mn.sup.++, Ca.sup.++ and
Zn.sup.++.
4. The method of claim 1 wherein said hybridization effective
amount of single-stranded nucleic acid binding protein is in the
range of 1 ng to 10 ug per 100 ng of template nucleic acid, and
said divalent cation is Mg.sup.++ present at a concentration of 1.5
mM.
5. A method of preparing a strand of DNA having a nucleotide
sequence substantially complementary to a template nucleic acid,
which method comprises:
(a) forming a primer extension reaction admixture whereby
nonspecific hybridization of polynucleotide synthesis is reduced by
admixing said template nucleic acid with a hybridization effective
amount of (i) a polynucleotide synthesis primer having a nucleotide
sequence complementary to a portion of the template nucleic acid,
(ii) isolated Eco SSB, (iii) Mg.sup.++, and (iv) a DNA polymerase;
and
(b) maintaining the primer extension reaction admixture under
polynucleotide synthesizing conditions for a time period sufficient
for said DNA polymerase to produce a primer extension product,
thereby producing said strand of DNA.
6. A method of amplifying a specific nucleic acid sequence present
in a sample containing a double-stranded nucleic acid molecule,
which method comprises:
(a) separating the strands of the double-stranded nucleic acid
molecule to form single-stranded templates;
(b) treating the single-stranded templates with hybridization
effective amounts of (i) primers that are selected so as to be
complementary to portions of the different strands of the specific
double-stranded sequence to hybridize therewith such that an
extension product synthesized from one primer, when it is separated
from its template nucleic acid strand, can serve as a template for
synthesis of the extension product of the other primer, (ii) an
isolated single-stranded nucleic acid binding protein (SSB) whereby
nonspecific hybridization of said primers is reduced, (iii) a
divalent cation, and (iv) an enzyme capable of producing primer
extension products from said single-stranded templates, wherein
said treatment is conducted under conditions such that the primers
hybridize to the specific sequences and an extension product of
each primer is synthesized by the action of the enzyme, said
product being substantially complementary to each single-stranded
template;
(c) separating the primer extension products formed in step (b)
from the templates on which they are synthesized to produce
single-stranded molecules; and
(d) treating the single-stranded molecules produced in step (c)
with hybridization effective amounts of the primers, SSB, divalent
cation and enzyme of step (b) under conditions such that a primer
extension product is synthesized using the single-stranded
molecules formed in step (c) as a template, thereby amplifying said
specific nucleic acid sequence.
7. The method of claim 6 wherein said single-stranded nucleic acid
binding protein is selected from the group consisting of Eco SSB,
T4 gene 32 protein and T7 SSB.
8. The method of claim 6 wherein said enzyme is a DNA polymerase
selected from the group consisting of Klenow fragment, Taq DNA
polymerase, recombinant Taq DNA polymerase, T7 DNA polymerase,
modified T7 DNA polymerase and T4 DNA polymerase.
9. The method of claim 6 wherein said divalent cation is selected
from the group consisting of Mg.sup.++, Mn.sup.++, Ca.sup.++ and
Zn.sup.++.
10. The method of claim 9 wherein said hybridization effective
amount of divalent cation is in the range of 0.5 mM to 20 mM.
11. The method of claim 9 wherein said divalent cation is Mg.sup.++
present at a concentration of about 1.5 mM.
12. The method of claim 6 wherein said hybridization effective
amount of single-stranded nucleic acid binding protein is in the
range of 1 ng to 10 ug per 100 ng of template nucleic acid.
13. The method of claim 6 wherein said separating in steps (a) and
(d) is accomplished by heat denaturing the double-stranded nucleic
acid molecule.
14. The method of claim 13 wherein said single-stranded nucleic
acid binding protein is heat stable.
15. The method claim 14 wherein said single-stranded nucleic acid
binding protein is Eco SSB.
16. The method of claim 13 wherein said DNA polymerase is a
heat-stable DNA polymerase.
17. The method of claim 16 wherein said heat-stable DNA polymerase
is Taq DNA polymerase or recombinant Taq DNA polymerase.
Description
TECHNICAL FIELD
The present invention relates to an improved process for producing
primed nucleic acid templates. More specifically, it relates to a
method of increasing the specificity of polynucleotide synthesis
via primer extension reactions by hybridizing primer to template in
the presence of a single-stranded nucleic acid binding protein.
BACKGROUND OF THE INVENTION
Primed templates are nucleic acids comprised of two nucleic acid
strands of unequal length bound together to form a substrate for
polynucleotide synthesis. Primed templates are used in a wide
variety of molecular biological techniques, including gene cloning,
in vitro gene mutagenesis, nucleic acid amplification, nucleic acid
detection, and the like.
Primed templates are typically produced by hybridizing (annealing)
a primer to a target nucleotide sequence on the template that is
complementary to the sequence of the primer. The fidelity of
hybridization reactions of primer with template is known to vary
depending on a variety of factors, including temperature,
complexity of the template, and the like. Generally, greater
template length and higher hybridization temperatures each
contribute to increased mismatching between primer and template
resulting in inappropriately and undesirably primed template.
Inappropriately primed template is the major cause of undesirable
(background) primer extension reaction products in primer extension
reactions. This is particularly the case in the polymerase chain
reaction (PCR) method for amplifying specific nucleic acid
sequences. See, for example, Beck et al, Nuc. Acid Res., 16: 9051
(1988); and Haqqi et al, Nuc. Acid Res., 16: 11844 (1988).
In PCR, specific nucleic acid sequences are amplified using a chain
reaction in which primer extension products are produced using
primed nucleic acid templates. The product of each primer extension
reaction specifically anneals with a primer and the resulting
primed template acts as a substrate for further primer extension
reactions. PCR is particularly useful in detecting nucleic acid
sequences which are initially present in only very small amounts.
However, the utility of PCR is often hampered by high levels of
background primer extension reaction products due to
primer/template mismatching. The procedure for conducting PCR has
been extensively described. See U.S. Pat. Nos. 4,683,195 and No.
4,683,202 both to Mullis et al.
Single-stranded nucleic acid binding proteins (SSB) have been
characterized in some detail and include such members as the E.
coli single-stranded binding protein (Eco SSB), T4 gene 32 protein
(T4 gp32), T4 gene 44/62 protein, T7 SSB, coliphage N4 SSB,
adenovirus DNA binding protein (Ad DBP or Ad SSB), and calf thymus
unwinding protein (UP1). Chase et al, Ann. Rev. Biochem., 55:
103-36 (1986); Coleman et al, CRC Critical Reviews in Biochemistry,
7(3): 247-289 (1980); Lindberg et al, J. Biol. Chem., 264:
12700-08, 1989; and Nakashima et al, FEBS Lett. 43: 125, 1974.
Eco SSB is an SSB that increases the fidelity of DNA replication
and stimulates E. coli DNA polymerases II and III but not
polymerase I or T4 DNA polymerase. Chase et al, Ann. Rev. Biochem.,
55: 103-36 (1986). Eco SSB has been shown to relieve pausing by DNA
polymerase III assemblies at regions of secondary structure
[(LaDuca et al, Biochem., 22: 5177-87 (1983)] and in vitro studies
of RecA-mediated reactions suggest that SSB affects ssDNA by
removing secondary structures. Muniyappa et al, Proc. Natl. Acad.
Sci. USA, 81: 2257-61 (1984).
T4 Gene 32 protein is an SSB coded for by gene 32 of bacteriophage
T4. One of its functions is to assist T4 DNA polymerase synthesis
across regions of secondary structure in a single-stranded
template. Huberman et al, J. Mol. Biol., 62: 39-52 (1971). It has
also been used in an in vitro mutagenesis reaction to promote
uninterrupted synthesis from template by addition of gene 32
protein to the template after a mutagenized primer is annealed but
before polymerization. Muta-Gene M13 in vitro mutagenesis kit
instructions, version #189 89-0096, p. 34, Bio-Rad Laboratories,
Richmond, Calif.
SSB therefore has traditionally been viewed as functioning by
minimizing secondary structure in ssDNA and thereby facilitating
enzyme passage (processivity) along the DNA template. In this
context, SSB has been used in a variety of ways based on this
property of SSB.
Zapoliski et al, in published PCT Patent Application No.
WO85/05685, describe the use of Eco SSB in combination with E. coli
RecA protein and ATP to form a hybridization mixture having the
capacity to stimulate the transfer of ssDNA to homologous duplexes.
The Zapoliski disclosure indicates that a cooperative binding
between Eco SSB and the ssDNA increases the homologous pairing
mediated by RecA.
Kaspar et al, Nuc. Acids Res., 17: 3616 (1989) describes the use of
T4 gp32 in the primer annealing and the primer extension steps of a
dsDNA sequencing procedure. The Kaspar disclosure states that
adding T4 gp32 allows Klenow enzyme to read through a region that
previously caused termination, which suggests that the utility of
T4 gp32 in a sequencing protocol was in reducing secondary
structure to allow the polymerase to continue down the
template.
BRIEF SUMMARY OF THE INVENTION
It has now been discovered that the presence of a single-stranded
nucleic acid binding protein (SSB) decreases the amount of
nonspecific primer extension products from primed templates.
This discovery has broad application to all procedures involving
the production of primed template by nucleic acid hybridization.
Particularly useful is the application of the discovery to methods
in which a specific nucleotide sequence is amplified, such as by
polymerase chain reaction, because the increased specificity of
primer to template hybridization makes the procedure unexpectedly
more sensitive.
Thus, the present invention contemplates a method of preparing a
primed template comprising the steps of (a) forming a nucleic acid
hybridization reaction admixture by admixing effective amounts of
(i) a primer nucleic acid capable of specifically hybridizing to a
template nucleic acid to form a substrate for polynucleotide
synthesis, (ii) the template nucleic acid, (iii) an isolated SSB,
and (iv) a divalent cation; and (b) maintaining said nucleic acid
hybridization reaction admixture under hybridizing conditions for a
time period sufficient for said primer nucleic acid to specifically
bind said template nucleic acid, thereby forming said primed
template. Preferably the SSB is Eco SSB, T4 gene 32 protein, or T7
SSB and more preferably is Eco SSB. It is also preferred that SSB
is present in an effective amount in the range of 1 ng to 10 ug per
100 ng of template nucleic acid. In addition, it is preferred that
the divalent cation be Mg.sup.++, Mn.sup.++, Ca.sup.++ or
Zn.sup.++, and more preferably Mg.sup.++ at an effective
concentration of 15 mM.
In another embodiment the invention contemplates a method of
preparing a strand of nucleic acid having a nucleotide sequence
complementary to a template nucleic acid comprising the steps of
(a) forming a primer extension reaction admixture by admixing the
template nucleic acid with an effective amount of (i) a
polynucleotide synthesis primer having a nucleotide sequence
substantially complementary to a portion of the template nucleic
acid, (ii) an isolated SSB, (iii) a divalent cation, and (iv) an
enzyme capable of inducing polynucleotide synthesis in the presence
of a primed template; and (b) maintaining the primer extension
reaction admixture under polynucleotide synthesizing conditions for
a time period sufficient for the enzyme to produce a primer
extension product, thereby producing the strand of nucleic acid.
Preferably the SSB is Eco SSB and the divalent cation is Mg.sup.++,
Mn.sup.++ Ca.sup.++ or Zn.sup.++.
Another embodiment contemplates a method of amplifying a specific
nucleic acid sequence present in a sample containing a
double-stranded nucleic acid molecule comprising the steps of: (a)
separating the strands of the double-stranded nucleic acid molecule
to form single-stranded templates; (b) treating the single-stranded
templates with the following in amounts effective to induce
polynucleotide synthesis under polynucleotide synthesizing
conditions: (i) primers that are selected so as to be substantially
complementary to portions of the different strands of the specific
double-stranded sequence to hybridize therewith such that an
extension product synthesized from one primer, when it is separated
from its template nucleic acid strand, can serve as a template for
synthesis of the extension product of the other primer, (ii) an
isolated SSB, (iii) a divalent cation, and (iv) an enzyme capable
of producing primer extension products from the single-stranded
templates, wherein the treatment is conducted under conditions such
that the primers hybridize to the specific sequences and an
extension product of each primer is synthesized by the action of
the enzyme, said product being complementary to each
single-stranded template; (c) separating the primer extension
products formed in step (b) from the templates on which they are
synthesized to produce single-stranded molecules; and (d) treating
the single-stranded molecules produced in step (c) with effective
amounts of the primers, SSB, divalent cation and enzyme of step (b)
under conditions such that a primer extension product is
synthesized using the single-stranded molecules formed in step (c)
as a template. Preferably the SSB is Eco SSB, T4 gene 32 protein,
or T7 SSB, and more preferably is the heat-stable SSB, Eco SSB. In
addition, a preferred enzyme is Klenow fragment, Taq DNA
polymerase, recombinant Taq DNA polymerase, T7 DNA polymerase,
modified T7 DNA polymerase or T4 DNA polymerase, and more
preferably is a heat-stable DNA polymerase such as Taq DNA
polymerase.
In another embodiment the invention contemplates a composition for
use in a polymerase chain reaction to amplify a specific nucleic
acid sequence comprising an isolated SSB, a divalent cation and two
polynucleotide primers that are selected so as to be sufficiently
complementary to different strands of a duplex DNA containing the
specific nucleic acid sequence as to hybridize therewith such that
an extension product synthesized from one primer, when it is
separated from its complementary nucleic acid strand, can serve as
a template for synthesis of the extension product of the other
primer, and wherein the SSB and primers are present in amounts
effective to support a polymerase chain reaction.
A related embodiment contemplates a composition for amplifying a
specific nucleic acid sequence in a double-stranded nucleic acid
form comprising the following components in an amount sufficient
for at least one assay: (a) an isolated SSB, (b) a divalent cation,
(c) a nucleic acid polymerase, and (d) two polynucleotide primers
that are selected so as to be sufficiently complementary to
different strands of the double stranded nucleic acid containing
the specific nucleic acid sequence to hybridize therewith such that
an extension product synthesized from one primer, when it is
separated from its complementary nucleic acid strand, can serve as
a template for synthesis of the extension product of the other
primer.
BRIEF DESCRIPTION OF THE DRAWINGS
In the drawings forming a portion of this disclosure:
FIG. 1 illustrates the sensitivity of a PCR amplification and
detection system for specific nucleic acids when conducted as
described in Example 2. Primer extension products produced by PCR
were analyzed by electrophoresis on 6% acrylamide gels, after
individual reaction mixtures were produced containing the following
amounts of lambda transgene mouse genomic DNA: 1 pg (lane 1), 10 pg
(lane 2), 100 pg (lane 3), 1 ng (lane 4) 10 ng (lane 5), 50 ng
(lane 6), 100 ng (lane 7) and 1 ug (lane 8). The arrow indicates
the position of the band representing an amplified nucleic acid
fragment that contains the specifically primed nucleic acid
sequence. All other bands are the result of non-specific primer
hybridization. Lane M contains molecular weight markers produced
from a Hinf I restriction endonuclease digest of bacteriophage phi
X174 DNA.
Panel A shows the results of a PCR amplification conducted in the
absence of Eco SSB. Panel B shows the results of a PCR
amplification under the same conditions as Panel A except that Eco
SSB was included in the hybridization and primer extension reaction
mixtures at 2.5 ug/ml.
FIG. 2 illustrates the range of effectiveness of an SSB protein for
improving a PCR-based specific nucleic acid detection system when
conducted as described in Example 2. Primer extension products were
produced in the presence of varying amounts of Eco SSB, and were
analyzed on 6% acrylamide gels as described in FIG. 1. The amount
of Eco SSB added per 100 ul of PCR mixture was: 10 ng (lane 1), 50
ng (lane 2), 100 ng (lane 3), 250 ng (lane 4), 500 ng (lane 5), 1
ug (lane 6), 2 ug (lane 7), 5 ug (lane 8), 10 ug (lane 9), and zero
ug (lane 10). The arrow indicates the position of the band
representing an amplified nucleic acid fragment that contains the
specifically primed nucleic acid sequence. All other bands are the
result of non-specific primer hybridization. Lane 9 lacks primer
extension products because of improper thermal cycling in that
reaction well of the thermal cycler apparatus, or because 10 ug of
Eco SSB may inhibit PCR.
FIG. 3 illustrates an analysis of hybridization stringency in a
PCR-based detection system when conducted as described in Example
2. Primer extension products produced by PCR using either a
42.degree. C. (lanes 1-6) or 54.degree. C. (lanes 7-12) annealing
temperature were analyzed as described in FIG. 1. PCR was conducted
in the presence (+) or absence (-) of 2.5 ug Eco SSB per ml PCR
mixture. Human genomic DNA was hybridized with primers pr 886 and
pr 887 (lanes 1, 2, 7 and 8), and lambda transgene mouse genomic
DNA (lanes 3-6 and lanes 9-12) was hybridized with an 18-mer primer
pair, pr 737 and pr 738 (lanes 3, 4, 9 and 10), or with a 23-mer
primer pair, pr 4266 and pr 19012 (lanes 5, 6, 11 and 12) in the
PCR method. The upper arrow indicates the position of the 1800 bp
specifically amplified nucleic acid fragment, and the lower arrow
indicates the position of the 520 bp specifically amplified nucleic
acid fragment. All other bands are the result of non-specific
primer hybridizations. Lane M contains the same molecular weight
markers as shown in FIG. 1.
FIG. 4 illustrates the use of two different primer pairs in a
PCR-based detection method, as described in Example 2, in which the
primers have regions of non-homology with the specific target
nucleic acid sequence. Primer extension products produced by PCR
using the primer pair pr 1A and pr 1B (lanes 1 and 2) or using the
primer pair pr 2A and pr 2B (lanes 3 and 4) were analyzed as
described in FIG. 1, and using the same markers in lane M. PCR was
conducted in the presence (lanes 2 and 4) or absence (lanes 1 and
3) of Eco SSB, and was directed at detecting the single copy DHFR
gene present in human genomic DNA. The arrow indicates the position
of the 250 bp band corresponding to a specifically amplified
nucleic acid fragment, which is visible in lanes 2, 3 and 4, but
not visible in lane 1.
DETAILED DESCRIPTION OF THE INVENTION
A. Definitions
Nucleotide: a monomeric unit of DNA or RNA consisting of a sugar
moiety (pentose), a phosphate, and a nitrogenous heterocyclic base.
The base is linked to the sugar moiety via the glycosidic carbon
(1' carbon of the pentose) and that combination of base and sugar
is a nucleoside. When the nucleoside contains a phosphate group
bonded to the 3' or 5' position of the pentose it is referred to as
a nucleotide. A sequence of operatively linked nucleotides is
typically referred to herein as a "base sequence" or "nucleotide
sequence", and is represented herein by a formula whose left to
right orientation is in the conventional direction of 5'-terminus
to 3'-terminus.
Duplex DNA: A double-stranded nucleic acid molecule comprising two
strands of substantially complementary polynucleotide hybridized
together by the formation of a hydrogen bond between each of the
complementary nucleotides present in a base pair of the duplex.
Because the nucleotides that form a base pair can be either a
ribonucleotide base or a deoxyribonucleotide base, the phrase
"duplex DNA" refers to either a DNA--DNA duplex comprising two DNA
strands (ds DNA), or an RNA-DNA duplex comprising one DNA and one
RNA strand.
Base Pair (bp): a partnership of adenine (A) with thymine (T), or
of cytosine (C) with guanine (G) in a double stranded DNA molecule.
In RNA, uracil (U) is substituted for thymine.
Nucleic Acid: a polymer of nucleotides, either single or double
stranded.
Gene: a nucleic acid whose nucleotide sequence codes for a RNA or
polypeptide. A gene can be either RNA or DNA.
Complementary Bases: nucleotides that normally pair up when DNA or
RNA adopts a double stranded configuration.
Complementary Nucleotide Sequence: a sequence of nucleotides in a
single-stranded molecule of DNA or RNA that is sufficiently
complementary to that on another single strand to specifically
(non-randomly) hybridize to it with consequent hydrogen
bonding.
Conserved: a nucleotide sequence is conserved with respect to a
preselected (reference) sequence if it non-randomly hybridizes to
an exact complement of the preselected sequence.
Hybridization: the pairing of complementary nucleotide sequences
(strands of nucleic acid) to form a duplex, heteroduplex or complex
containing more than two single-stranded nucleic acids by the
establishment of hydrogen bonds between/among complementary base
pairs. It is a specific, i.e. non-random, interaction between/among
complementary polynucleotides that can be competitively
inhibited.
Hybridization product: The product formed when a polynucleotide
hybridizes to a single or double-stranded nucleic acid. When a
polynucleotide hybridizes to a double-stranded nucleic acid, the
hybridization product formed is referred to as a triple helix or
triple-stranded nucleic acid molecule. Moser et al, Science, 238:
645-50 (1987).
Nucleotide Analog: a purine or pyrimidine nucleotide that differs
structurally from an A, T, G, C, or U, but is sufficiently similar
to substitute for the normal nucleotide in a nucleic acid molecule.
Inosine (I) is a nucleotide that can hydrogen bond with any of the
other nucleotides, A, T, G, C, or U. In addition, methylated bases
are known that can participate in nucleic acid hybridization.
B. Methods
The present invention broadly contemplates an improvement to well
known nucleic acid hybridization methods. The improvement comprises
the use of SSB in the hybridization reaction admixtures to decrease
the amount of nonspecific hybridization between complementary
nucleic acids.
Because of the basic nature of the hybridization reaction in
recombinant nucleic acid technologies, it is contemplated that the
improvement can be practiced on other methods not specifically
described herein that utilize hybridization as a part of the
method. Therefore the following discussion is not intended to limit
the present invention, but is provided to illustrate the
application of principles that can be relied on to improve nucleic
acid hybridization methods in general.
1. Single-stranded Nucleic Acid Binding Protein
In all embodiments described herein there is a single-stranded
nucleic acid binding protein (SSB) included in the hybridization
reaction admixture.
"Single-stranded nucleic acid binding protein" as used herein
refers to a class of proteins collectively referred to by the term
SSB. Chase et al, Ann. Rev. Biochem., 55: 103-36 (1986). (The art
cited herein is hereby incorporated by reference.) SSB has the
general property of preferential binding to single-stranded (ss)
over double-stranded (ds) nucleic acids irrespective of the
nucleotide sequence. The class includes such diverse members as Eco
SSB, T4 gp32, T7 SSB, N4 SSB, Ad SSB, UP1, and the like. Chase et
al, Ann. Rev. Biochem., 55: 103-36 (1986); and Coleman et al, CRC
Critical Reviews in Biochemistry, 7(3): 247-289 (1980).
SSB has a strong binding affinity for ssRNA almost as well as
ssDNA, and binds dsDNA substantially less well. For example, Eco
SSB in general binds ssDNA ten times better than ssRNA, and
10.sup.3 times better than dsDNA. Chase et al, Ann. Rev. Biochem;
55: 103-36 (1986).
A preferred SSB for use in the methods disclosed herein is Eco SSB
or T4 gp32. More preferred and exemplary of the disclosed methods
is Eco SSB, whose preparation and use is described herein.
SSB has other general properties described by Chase et al, Ann.
Rev. Biochem., 55: 103-36 (1986). These properties include the
ability of an SSB to reduce the melting temperature of dsDNA, to
increase processivity of an accessory DNA polymerase, to bind ssDNA
stoichiometrically in an amount that depends on the particular SSB
protein, and to destablize secondary structures in ssDNA.
The known SSBs all exhibit the characteristic of increasing the
processivity of their accessory DNA polymerases. An SSB is deemed
accessory to a DNA polymerase when both the SSB and the DNA
polymerase are present in the same organism and characterized as
having a capacity to stimulate a particular DNA polymerase. For
example, Eco SSB is accessory to E. coli. DNA polymerase II and III
because it stimulates that polymerase's activity, but does not
stimulate T4 DNA polymerase.
Other SSBs useful in practicing the present invention are modified
in amino acid residue sequence in some degree as to retain the
general properties as described above. For example the protein
produced by the mutant allele ssb-113 in E. coli. produces an SSB
that binds to ssDNA as well as wild-type Eco SSB but exhibits a
greater capacity to lower the melting temperature of dsDNA. Chase
et al, J. Biol. Chem., 259: 805-14 (1984). Therefore "modified"
SSBs, derived by isolation of mutants or by manipulation of cloned
SSB protein-encoding genes, are also contemplated for use in the
presently disclosed methods.
An SSB as disclosed herein can be used alone or in combination with
other SSBs in the disclosed methods.
An effective amount of one or more SSBs for use in the disclosed
methods depends on the amount of nucleic acid present in the
admixtures, as it is known that SSB binds to nucleic acids
stoichiometrically. For example, Eco SSB binds ssDNA to a maximum
of about one SSB binding site per 33 to 65 base nucleotides,
depending upon the salt concentration. Lohman et al, J. Biol.
Chem., 260: 3594-603 (1985). Therefore, an effective amount of SSB
is typically in the range of 1 ng to 10 ug of SSB protein per 100
ng of nucleic acid, preferably about 0.1 ug to 5 ug, and more
preferably about 0.25 ug to 2 ug of SSB protein per 100 ng nucleic
acid.
To be effective in the presently contemplated methods, SSB must be
admixed with a nucleic acid in the presence of an activating
(effective) amount of divalent cations. Preferred divalent cations
for activating SSB are Mg.sup.++, Mn.sup.++, Ca.sup.++ and
Zn.sup.++. More preferably, Mg.sup.++ is used to activate SSB. An
activating amount of divalent cations is in the range of 100 uM to
30 mM, and preferably 500 uM to 20 mM, more preferably about 15 mM
for hybridization reactions. Christiansen et al, J. Mol. Biol.,
115: 441-54 (1977). When SSB is used in a PCR-based method it is
preferred that the divalent cations be present in the range of 1 to
5 mM, and more preferably the divalent cation is Mg.sup.++ used at
about 1.5 mM.
SSBs can be isolated from their source organisms by standard
biochemical methods involving cell lysis and protein
chromatography. Particularly preferred are the methods described in
Example 1 including ammonium sulfate and polyethylenimine
fractionation, affinity chromatography on ssDNA-cellulose and
phosphocellulose chromatography. Alternatively, several of the SSBs
are commercially available.
Eco SSB can be obtained commercially from Pharmacia, Inc.
(Piscataway, N.J.), or can be prepared from a variety of E. coli
strains, such as is described by Chase et al, Nuc. Acid Res., 8:
3215-27 (1980), or by Weiner et al, J. Biol. Chem., 250: 1972-80
(1975), or can purified by the method disclosed in Example 1 using
any of a variety of E. coli strains, including E. coli B or E. coli
strain K12-H1-TRP containing the plasmid pTL119A-5.
T4 gp32 can be obtained commercially from Pharmacia, Bio-Rad
(Richmond, Calif.) or Boeringer Mannheim Biochemicals
(Indianapolis, Ind.), or can be prepared from a T4 infected
bacterial culture such as is described by Jarvis et al, J. Biol.
Chem., 264: 12709-16 (1989).
Because SSB is utilized in the methods of this invention in
admixture with nucleic acids, it is preferred that SSB be
substantially free of nuclease activity. By "substantially free of
nuclease activity" is meant that on admixture and incubation of an
SSB preparation with target nucleic acid molecules under
hybridization reaction conditions, more than 70 percent of the
target nucleic acid molecules, and preferably more than 95 percent
of the target molecules, retain the capacity to hybridize with
complementary polynucleotide molecules when measured by a
hybridization procedure such as is described in Example 2. SSB
substantially free of nuclease activity (isolated SSB) can be
prepared by the methods described in Example 1, or can be obtained
from the commercial vendors described above.
Where SSB is to be utilized in a hybridization method in which a
hybridization reaction admixture containing SSB is subjected to a
temperature in the range of about 30 degrees centigrade (30.degree.
C.) to about 100.degree. C., it is preferred that the SSB is a
heat-stable SSB. A "heat-stable SSB" as defined herein is an SSB
that retains more than 90% of its pre-treatment capacity to bind
ssDNA preferentially over dsDNA irrespective of the nucleotide
sequence of the ssDNA after exposure in a hybridization reaction
admixture to 90 degrees C. (90.degree. C.) for 10 min, preferably
after exposure to 95.degree. C. for 10 min, and more preferably
after exposure to 100.degree. C. for 8 min. A preferred heat-stable
SSB is Eco SSB, that retains about 90% of its capacity to bind
ssDNA after exposure to 100.degree. C. for 8 min. Weiner et al, J.
Biol. Chem., 250: 1972-80 (1975).
2. Preparation of Polynucleotides
The term "polynucleotide" as used herein refers to a nucleic acid
molecule comprised of a linear strand of two or more
deoxyribonucleotides and/or ribonucleotides, preferable more than
3. The exact size will depend on many factors, which in turn
depends on the ultimate conditions of use, as is well known in the
art. Polynucleotides used in the present invention include primers,
probes, and the like.
The term "primer" as used herein refers to a polynucleotide,
whether purified from a nucleic acid restriction digest or produced
synthetically, which is capable of acting as a point of initiation
of synthesis when placed under conditions in which synthesis of a
primer extension product which is complementary to a template
nucleic acid strand is induced, i.e., in the presence of
nucleotides and an agent for polymerization such as DNA polymerase,
reverse transcriptase and the like, and at a suitable temperature
and pH.
The primer must be sufficiently long to prime the synthesis of
extension products in the presence of the agents for
polymerization. The exact lengths of the primers will depend on
many factors, including temperature and the source of primer. For
example, depending on the complexity of the template sequence, a
polynucleotide primer typically contains 15 to 25 or more
nucleotides, although it can contain fewer nucleotides. As few as 8
nucleotides in a polynucleotide primer have been reported as
effective for use. Studier et al, Proc. Natl. Acad. Sci. USA, 86:
6917-21 (1989). Short primer molecules generally require lower
temperatures to form sufficiently stable hybridization complexes
with template to initiate primer extension.
The primers used herein are selected to be "substantially"
complementary to the different strands of each specific sequence to
be synthesized or amplified. This means that the primer must
contain at its 3' terminus a nucleotide sequence sufficiently
complementary to nonrandomly hybridize with its respective template
strand. Therefore, the primer sequence may not reflect the exact
sequence of the template. For example, a non-complementary
polynucleotide can be attached to the 5' end of the primer, with
the remainder of the primer sequence being substantially
complementary to the strand. Such noncomplementary polynucleotides
might code for an endonuclease restriction site or a site for
protein binding. Alternatively, noncomplementarity bases or longer
sequences can be interspersed into the primer, provided the primer
sequence has sufficient complementarity with the sequence of the
strand to be synthesized or amplified to non-randomly hybridize
therewith and thereby form an extension product under
polynucleotide synthesizing conditions.
Sommer et al, Nuc. Acid Res., 17: 6749 (1989), reports that primers
having as little as a 3 nucleotide exact match at the 3' end of the
primer were capable of specifically initiating primer extension
products, although less nonspecific hybridization occurs when the
primer contains more nucleotides at the 3' end having exact
complementarity with the template sequence. Therefore, a
substantially complementary primer as used herein must contain at
its 3' end at least 3 nucleotides having exact complementarity to
the template sequence. A substantially complementary primer
preferably contains at least 10 nucleotides, more preferably at
least 18 nucleotides, and still more preferably at least 24
nucleotides, at its 3' end having the aforementioned
complementarity. Still more preferred are primers whose entire
nucleotide sequence have exact complementarity with the template
sequence.
The choice of a primer's nucleotide sequence depends on factors
such as the distance on the nucleic acid from the region coding for
the desired specific nucleic acid sequence present in a nucleic
acid of interest and its hybridization site on the nucleic acid
relative to any second primer to be used.
The primer is preferably provided in single-stranded form for
maximum efficiency, but may alternatively be double stranded. If
double stranded, the primer is first treated to separate its
strands before being used to prepare extension products.
Preferably, the primer is a polydeoxyribonucleotide.
Polynucleotides can be prepared by a variety of methods including
de novo chemical synthesis of polynucleotides and derivation of
nucleic acid fragments from native nucleic acid sequences existing
as genes, or parts of genes, in a genome, plasmid, or other vector,
such as by restriction endonuclease digest of larger
double-stranded nucleic acids and strand separation or by enzymatic
synthesis using a nucleic acid template.
De novo chemical synthesis of a polynucleotide can be conducted
using any suitable method, such as, for example, the
phosphotriester or phosphodiester methods. See Narang et al, Meth.
Enzymol., 68: 90, (1979); U.S. Pat. No. 4,356,270; Itakura et al,
Ann. Rev. Biochem., 53: 323-56 (1989); and Brown et al, Meth.
Enzymol., 68: 109, (1979).
Derivation of a polynucleotide from nucleic acids involves the
cloning of a nucleic acid into an appropriate host by means of a
cloning vector, replication of the vector and therefore
multiplication of the amount of the cloned nucleic acid, and then
the isolation of subfragments of the cloned nucleic acids. For a
description of subcloning nucleic acid fragments, see Maniatis et
al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory, pp 390-401 (1982); and see U.S. Pat. Nos. 4,416,988 and
No. 4,403,036.
3. Preparing Primed Template
In one embodiment, the present invention contemplates an improved
method for hybridizing a primer to a template sequence present on a
nucleic acid to form a primer-template hybridization product
(primed template). A hybridization reaction admixture is prepared
by admixing effective amounts of a primer, a template nucleic acid
and other components compatible with a hybridization reaction
including an effective amount of a single-stranded nucleic acid
binding protein (SSB).
The primary purpose in including SSB in the hybridization reaction
admixture is to reduce nonspecific hybridization reactions. Because
nonspecific hybridization occurs more frequently as the complexity
of the template nucleic acid increases, the benefits of including
SSB in primer-template hybridization reaction admixtures increase
as the complexity of the template increases. Thus, the method of
the present invention is particularly useful where the template
nucleic acid has a complexity comparable to that of lambda
bacteriophage DNA. The benefits are even more pronounced where the
complexity of template is comparable to a procaryotic or eucaryotic
genome.
Template nucleic acid sequences to be hybridized in the present
methods can be present in any nucleic acid-containing sample so
long as the sample is in a form, with respect to purity and
concentration, compatible with nucleic acid hybridization reaction.
Isolation of nucleic acids to a degree suitable for hybridization
is generally known and can be accomplished by a variety of means.
For instance, nucleic acids can be isolated from a variety of
nucleic acid-containing samples including body tissue, such as
skin, muscle, hair, and the like, and body fluids such as blood,
plasma, urine, amniotic fluids, cerebral spinal fluids, and the
like. See, for example, Maniatis et al, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory (1982); and
Ausubel et al, Current Protocols in Molecules Biology, John Wiley
and Sons (1987).
The hybridization reaction mixture is maintained under hybridizing
conditions for a time period sufficient for the primer to hybridize
to complementary nucleic acid sequences present in the sample to
form a hybridization product, i.e., a complex containing primer and
template nucleic acid strands.
The phrase "hybridizing conditions" and its grammatical
equivalents, when used with a maintenance time period, indicates
subjecting the hybridization reaction admixture, in the context of
the concentrations of reactants and accompanying reagents in the
admixture, to time, temperature and pH conditions sufficient to
allow the primer to anneal with the template sequence, typically to
form a nucleic acid duplex. Such time, temperature and pH
conditions required to accomplish hybridization depend, as is well
known in the art, on the length of the primer to be hybridized, the
degree of complementarity between the primer and the template, the
guanidine and cytosine content of the polynucleotide, the
stringency of hybridization desired, and the presence of salts or
additional reagents in the hybridization reaction admixture as may
affect the kinetics of hybridization. Methods for optimizing
hybridization conditions for a given hybridization reaction
admixture are well known in the art.
Typical hybridizing conditions include the use of solutions
buffered to pH values between 4 and 9, and are carried out at
temperatures from 18 degrees C (18.degree. C.) to 75.degree. C.,
preferably about 37.degree. C. to about 65.degree. C., more
preferably about 54.degree. C., and for time periods from 0.5
seconds to 24 hours, preferably 2 min.
Hybridization can be carried out in a homogeneous or heterogeneous
format as is well known. The homogeneous hybridization reaction
occurs entirely in solution, in which both the primer and template
sequences to be hybridized are present in soluble forms in
solution. A heterogeneous reaction involves the use of a matrix
that is insoluble in the reaction medium to which either the primer
or template is bound.
Also preferred are the homogeneous hybridization reactions such as
are conducted for a reverse transcription of isolated mRNA to form
CDNA, dideoxy sequencing and other procedures using primer
extension reactions in which primer hybridization is a first step.
Particularly preferred is the homogeneous hybridization reaction in
which the template is amplified via a polymerase chain reaction
(PCR).
The use of a SSB in a hybridization reaction admixture reduces the
degree of nonspecific hybridization of a primer to a template
nucleic acid. Therefore hybridization conditions can be carried out
at temperatures lower than normally required to maintain high
stringency, i.e., high specificity, in the hybridization
reaction.
Where the nucleic acid containing a template sequence is in a
double-stranded (ds) form, it is preferred to first denature the
dsDNA, as by heating or alkali treatment, prior to conducting the
hybridization reaction. The denaturation of the dsDNA can be
carried out prior to admixture with a primer to be hybridized, or
it can be carried out after the admixture of the dsDNA with the
primer, the SSB or both. Where the primer itself is provided as a
double-stranded molecule, it too can be denatured prior to
admixture, or it can be denatured concurrently with the
template-containing dsDNA.
4. Primer Extension Reactions
The primed template can be used to produce a strand of nucleic acid
having a nucleotide sequence complementary to the template, i.e., a
template-complement.
If the template whose complement is to be produced is in the form
of double stranded nucleic acid, it is typically first denatured,
usually by melting, into single strands, such as ssDNA. The nucleic
acid is then subjected to a (first) primer extension reaction by
treating (contacting) the nucleic acid with a (first)
polynucleotide synthesis primer having as a portion of its
nucleotide sequence a sequence selected to be substantially
complementary to a portion of the sequence of the template. The
primer is capable of initiating a primer extension reaction by
hybridizing to a specific nucleotide sequence, preferably at least
about 8 nucleotides in length and more preferably at least about 20
nucleotides in length. This is accomplished by mixing an effective
amount of the primer with the template nucleic acid, an effective
amount of SSB, and an effective amount of nucleic acid synthesis
inducing agent to form a primer extension reaction admixture. The
admixture is maintained under polynucleotide synthesizing
conditions for a time period, which is typically predetermined,
sufficient for the formation of a primer extension reaction
product.
The primer extension reaction is performed using any suitable
method. Generally polynucleotide synthesizing conditions are those
wherein the reaction occurs in a buffered aqueous solution,
preferably at a pH of 7-9, most preferably about 8. Preferably, a
molar excess (for genomic nucleic acid, usually about 10.sup.6 :1
primer:template) of the primer is admixed to the buffer containing
the template strand. A large molar excess is preferred to improve
the efficiency of the process. For polynucleotide primers of about
20 to 25 nucleotides in length, a typical ratio is in the range of
50 ng to 1 ug, preferably 250 ng, of primer per 100 ng to 500 ng of
mammalian genomic DNA or per 10 to 50 ng of plasmid DNA.
The deoxyribonucleotide triphosphates (dNTPs) DATP, dCTP, dGTP, and
dTTP are also admixed to the primer extension reaction admixture in
amounts adequate to support the synthesis of primer extension
products, and depends on the size and number of products to be
synthesized. The resulting solution is heated to about 90.degree.
C.-100.degree. C. for about 1 to 10 minutes, preferably from 1 to 4
minutes. After this heating period the solution is allowed to cool
to room temperature, which is preferable for primer hybridization.
To the cooled mixture is added an appropriate agent for inducing or
catalyzing the primer extension reaction, and the reaction is
allowed to occur under conditions known in the art. The synthesis
reaction may occur at from room temperature up to a temperature
above which the inducing agent no longer functions efficiently.
Thus, for example, if DNA polymerase is used as inducing agent, the
temperature is generally no greater than about 40.degree. C. unless
the polymerase is heat-stable.
The inducing agent may be any compound or system which will
function to accomplish the synthesis of primer extension products,
including enzymes. Suitable enzymes for this purpose include, for
example, E. coli, DNA polymerase I, Klenow fragment of E. coli DNA
polymerase I, T4 DNA polymerase, T7 DNA polymerase, recombinant
modified T7 DNA polymerase, other available DNA polymerases,
reverse transcriptase, and other enzymes, including heat-stable
enzymes, which will facilitate combination of the nucleotides in
the proper manner to form the primer extension products which are
complementary to each nucleic acid strand.
Heat-stable DNA polymerases are particularly preferred as they are
stable in a most preferred embodiment in which PCR is conducted in
a single solution in which the temperature is cycled.
Representative heat-stable polymerases are the DNA polymerases
isolated from Bacillus stearothermophilus (Bio-Rad), Thermus
thermophilus (FINZYME, ATCC #27634), Thermus species (ATCC #31674),
Thermus aquaticus strain TV 1151B (ATCC #25105), Sulfolobus
acidocaldarius, described by Bukhrashuili et al, Biochem. Biophys.
Acta, 1008: 102-7 (1989) and by Elie et al, Biochem. Biophys. Actz,
951: 261-7 (1988), and Thermus filiformis (ATCC #43280).
Particularly preferred is Taq DNA polymerase available from a
variety of sources including Perkin Elmer Cetus, (Norwalk, Conn.),
Promega (Madison, Wis.) and Stratagene (La Jolla, Calif.), and
AmpliTaq.TM. DNA polymerase, a recombinant Taq DNA polymerase
available from Perkin-Elmer Cetus.
Generally, the synthesis will be initiated at the 3' end of each
primer and proceed in the 5' direction along the template strand,
until synthesis terminates, producing molecules of different
lengths. There may be inducing agents, however, which initiate
synthesis at the 5' end and proceed in the above direction, using
the same process as described above.
The primer extension reaction product can then be subjected to a
second primer extension reaction by treating it with a second
polynucleotide synthesis primer having a preselected nucleotide
sequence. The second primer is capable of initiating the second
reaction by hybridizing to a nucleotide sequence, preferably at
least about 8 nucleotides in length and more preferably at least
about 20 nucleotides in length, found in the first product. This is
accomplished by mixing the second primer, preferably a
predetermined amount thereof, with the first product, preferably a
predetermined amount thereof, to form a second primer extension
reaction admixture. The admixture is maintained under
polynucleotide synthesizing conditions for a time period, which is
typically predetermined, sufficient for the formation of a second
primer extension reaction product.
In preferred strategies, the first and second primer extension
reactions are the first and second primer extension reactions in a
polymerase chain reaction (PCR).
PCR is carried out by simultaneously cycling, i.e., performing in
one admixture, the above described first and second primer
extension reactions, each cycle comprising polynucleotide synthesis
followed by denaturation of the double stranded polynucleotides
formed. Methods and systems for amplifying a specific nucleic acid
sequence are described in U.S. Pat. Nos. 4,683,195 and No.
4,683,202, both to Mullis et al; and the teachings in PCR
Technology, Erlich, ed., Stockton Press (1989); Faloona et al,
Methods in Enzymol., 155: 335-50 (1987); and Polymerase Chain
Reaction, Erlich et al, eds., Cold Spring Harbor Laboratories Press
(1989).
In one embodiment, a method of amplifying a specific
double-stranded nucleic acid sequence in a nucleic acid sample is
contemplated. The method comprises the steps of:
(a) Separating the strands of the double-stranded nucleic acid
molecule to form single-stranded templates.
(b) Treating the single-stranded templates with the following in
amounts effective to induce polynucleotide synthesis under
polynucleotide synthesizing conditions: (i) primers that are
selected so as to be substantially complementary to portions of the
different strands of the specific double-stranded sequence to
hybridize therewith such that an extension product synthesized from
one primer, when it is separated from its template (complementary)
nucleic acid strand, can serve as a template for synthesis of the
extension product of the other primer, (ii) an SSB, preferably Eco
SSB, and (iii) an enzyme capable of producing primer extension
products from said single-stranded templates, wherein said
treatment is conducted under conditions such that the primers
hybridize to the specific sequences and an extension product of
each primer is synthesized by the action of the enzyme, said
product being complementary to each single-stranded template.
(c) Separating the primer extension products formed in step (b)
from the templates on which they are synthesized to produce
single-stranded molecules.
(d) Treating the single-stranded molecules produced in step (c)
with effective amounts of the primers, SSB and enzyme of step (b)
under conditions such that a primer extension product is
synthesized using the single-stranded molecules formed in step (c)
as a template.
PCR is carried out by cycling i.e., simultaneously performing in
one admixture, the above described: 1) denaturing step to form
single-stranded templates, 2) hybridization step to hybridize
primer to ss template, and 3) primer extension steps to form the
extended product. PCR is performed in the above sequence (cycle) by
changing the temperature of the PCR mixture to a temperature
compatible with each step, in series.
The primer extension reaction conditions involve maintaining the
reaction mixture for a time period and at a temperature sufficient
for a DNA polymerase primer extension reaction to occur to produce
primer extension products as is well known. Conditions for
conducting a primer extension reaction are well known. In a PCR
format, the maintenance is carried out quickly to conveniently
facilitate numerous cycles, in about 1 second to 5 minutes,
preferably about 1.5 minutes, and at about 40.degree. C. to
75.degree. C., preferably about 72.degree. C. Conducting at least
one cycle of PCR results in the formation of amplified nucleic acid
products. The PCR is typically conducted with at least 15 cycles,
and preferably with about 20 to 40 cycles.
Hybridizing conditions were described earlier and are suitable for
use in the PCR format. However, it is preferred and convenient to
conduct hybridization in short periods of time, in 5 seconds to 12
minutes, preferably in 2 minutes, and in the temperature range of
30.degree. C. to 75.degree. C., preferably about 40.degree. C. to
65.degree. C., and more preferably about 54.degree. C.
A suitable SSB for use in a hybridization reaction mixture is one
of the class of SSBs described previously, preferably a nuclease
free, heat-stable SSB, and more preferably is Eco SSB. Exemplary
hybridization reaction mixtures and reaction conditions are
described further in Example 2.
In preferred embodiments, detecting the presence of any primer
extension product formed in step (d) thereby provides a method for
detecting the presence of a specific double-stranded nucleic acid
sequence in the sample.
Detection of amplified nucleic acid product can be accomplished by
any of a variety of well known techniques. In a preferred
embodiment, the amplified product is separated on the basis of
molecular weight by gel electrophoresis, and the separated products
are then visualized by the use of nucleic acid specific stains
which allow one to observe the discreet species of resolved
amplified product present in the gel. Although numerous nucleic
acid specific stains exist and would be suitable to visualize the
electrophoretically separated nucleic acids, ethidium bromide is
preferred.
Alternative methods suitable to detect the amplified nucleic acid
product include hybridization-based detection means that use a
labeled polynucleotide probe capable of hybridizing to the
amplified product. Exemplary of such detection means include the
Southern blot analysis, ribonuclease protection analysis using in
vitro labeled polyribonucleotide probes, and the like methods for
detecting nucleic acids having specific nucleotide sequences. See,
for example, Ausubel et al., Current Protocols in Molecular
Biology, John Wiley & Sons, 1987.
In one approach for detecting the presence of a specific nucleic
acid sequence, the deoxyribonucleotide triphosphates (dNTPs) used
in the primer extension reaction include a label or indicating
group that will render a primer extension product detectable.
Typically such labels include radioactive atoms, chemically
modified nucleotide bases, and the like.
Radioactive elements operatively linked to or present as part of a
DNTP provide a useful means to facilitate the detection of a DNA
primer extension product. A typical radioactive element is one that
produces beta ray emissions. Elements that emit beta rays, such as
.sup.3 H, .sup.14 C, .sup.32 p, and .sup.35 S represent a class of
beta ray emission-producing radioactive element labels.
Alternatives to radioactively labeled dNTPs are dNTPs that are
chemically modified to contain metal complexing agents,
biotin-containing groups, fluorescent compounds, and the like.
One useful metal complexing agent is a lanthanide chelate formed by
a lanthanide and an aromatic beta-diketone, the lanthanide being
bound to the nucleic acid or polynucleotide via a chelate forming
compound such as an EDTA-analogue so that a fluorescent lanthanide
complex is formed. See U.S. Pat. Nos. 4,374,120, and No. 4,569,790
and published International Patent Applications No. EP0139675 and
No. WO87/02708.
Biotin or acridine ester-labeled oligonucleotides and their use in
polynucleotides have been described. See U.S. Pat. No. 4,707,404,
published International Patent Application EP0212951 and European
Patent No. 0087636. Useful fluorescent marker compounds include
fluorescein, rhodamine, Texas Red, NBD and the like.
A labeled nucleotide residue present in a nucleic acid renders the
nucleic acid itself labeled and therefore distinguishable over
other nucleic acids present in a sample to be assayed. Detecting
the presence of the label in the nucleic acid and thereby the
presence of the specific nucleic sequence, typically involves
separating the nucleic acid from any labeled dNTP that is not
present as part of a primer extension reaction product.
Numerous applications of the PCR-based amplification method are
contemplated that will be readily apparent to one skilled in the
art. For example, cloning mRNA through reverse transcription to
produce CDNA can be made more sensitive by the use of PCR-based
amplification of the produced cDNA. Insofar as nucleic acid
sequencing can be conducted on PCR-amplified nucleic acid, the
present invention can be used to improve the amplified nucleic
acids production step in a sequencing procedure. A variety of the
other recombinant DNA cloning and nucleic acid manipulative steps
have been described that involved PCR. See, for example, PCR
Technology, Erlich, ed., Stockton Press (1989), and Polymerase
Chain Technology, Erlich et al, eds., Cold Spring Harbor Laboratory
Press (1989).
In a related embodiment, the present invention contemplates the
application of the improved PCR amplification method to samples of
nucleic acid in which the nucleic acid is hybridized and primer
extension products are formed from a hybridization product
comprised of triple-stranded nucleic acid molecules.
Triple-stranded nucleic acid molecules, or triple helixes, have
been produced in vitro by hybridization of polynucleotides and
duplex DNA. Moser et al, Science, 238: 645-50 (1987); Kohwi et al,
Proc. Natl. Acad. Sci. USA, 85: 3781-85 (1988); Wells et al, FASEB
J., 2: 2939-49 (1988); Htun et al, Science, 243: 1571-76 (1989);
and Rajagopal et al, Nature, 339: 637-40 (1989). Therefore although
a preferred template for hybridization and primer extension in a
PCR method is a single-stranded nucleic acid, the invention
contemplates the use of a double-stranded template to form a
triple-stranded hybridization product.
In practicing the methods of the present invention to form a
triple-stranded hybridization product, it is to be understood that
the initial denaturation of double-stranded nucleic acids to form
single-stranded template is not required. Instead, the
hybridization reaction mixture can be used to treat double-stranded
template rather than single-stranded template forming a triple
helix hybridization product.
C. Compositions
Also contemplated by the present invention is a composition for
producing a template-complement from a nucleic acid template that
contains a specific nucleic acid sequence according to the methods
of the present invention. The composition comprises, in amounts
sufficient to produce at least one template-complement, (a) an
isolated single-stranded nucleic acid binding protein, and (b) a
polynucleotide primer selected so as to be sufficiently
complementary to a portion of a nucleic acid template containing
said specific nucleic acid sequence to hybridize therewith such
that a primer extension product can be synthesized therefrom in a
primer extension reaction.
In another embodiment, a composition for producing a
template-complement comprises effective amounts of (a) an isolated
single-stranded nucleic acid binding protein, and (b) an inducing
agent for nucleic acid synthesis by primer extension. A preferred
inducing agent is one of the previously described DNA polymerase
enzymes suitable for the synthesis of primer extension
products.
In one embodiment for amplifying a specific nucleic acid via the
PCR, composition comprises, in effective amounts, (a) an isolated
single-stranded nucleic acid binding protein, and (b) two
polynucleotide primers that are selected so as to be substantially
complementary to portions of different strands of a double-stranded
nucleic acid containing said specific nucleic acid sequence to
hybridize therewith such that an extension product synthesized from
one primer, when it is separated from its complementary nucleic
acid strand, can serve as a template for synthesis of the extension
product of the other primer.
In all embodiments of a contemplated composition additional
reagents can also be included such as a divalent cation, preferably
Mg.sup.++, Mn.sup.++, Ca.sup.++, or Zn.sup.++, and preferably an
amount of divalent cation sufficient to produce a primer extension
reaction mixture having an effective amount of divalent cation
concentration in the range of 0.5 mM to 20 mM. Where the
composition is formulated for use in PCR-based methods, it is
preferred that the divalent cation is Mg.sup.++ and the effective
amount is about 1.5 mM.
The single-stranded binding protein (SSB) for inclusion in a
composition of this invention is one of the SSBs described herein
for practicing a contemplated method. The amount of SSB to be
included in a system is an amount sufficient to produce a reaction
mixture having an effective amount of SSB in the range of 1 ng to
10 ng per 100 ng of nucleic acid, and more preferably is about 0.25
ug to 2 ug of SSB per 100 ng of nucleic acid.
A particularly preferred composition for use in PCR includes
isolated Eco SSB, Mg.sup.++ in an amount sufficient to produce a
reaction mixture concentration of about 1.5 mM, and a heat-stable
DNA polymerase, preferably Taq polymerase. An additional reagent
for this embodiment can be two polynucleotide primers selected
having the capacity to function in PCR as described above.
The compositions can be packaged in kit form. As used herein, the
term "package" refers to a solid matrix or material customarily
utilized in a system and capable of holding within fixed limits one
or more of the reagent components for use in a method of the
present invention. Such materials include glass and plastic (e.g.,
polyethylene, polypropylene and polycarbonate) bottles, vials,
paper, plastic and plastic-foil laminated envelopes and the like.
Thus, for example, a package can be a glass vial used to contain
the appropriate quantities of polynucleotide primer(s), isolated
SSB, a divalent cation and a DNA polymerase, or a combination
thereof.
In embodiments where the system is utilized diagnostically to
detect the presence of a specific nucleic acid, it is preferred
that the diagnostic system further includes a label or indicating
means capable of signaling the formation of a complex containing a
polynucleotide by the methods of the present invention.
In preferred embodiments, the indicating means comprises a probe,
the probe being operatively linked to a label, thereby providing a
means to detect an amplified nucleic acid product formed. Preferred
labels are those discussed hereinbefore, especially .sup.32 p and
.sup.35 S for polynucleotide probes.
Kits useful for producing a template-complement or for
amplification or detection of a specific nucleic acid sequence
using a primer extension reaction methodology also typically
include, in separate containers within the kit, dNTPs where N is
adenine, thymine, guanine and cytosine, and other like agents for
performing primer extension reactions.
The reagent species, indicating means or primer extension reaction
reagents of any system described herein can be provided in
solution, as a liquid dispersion or as a substantially dry power,
e.g., in lyophilized form. Where the reagent species or indicating
means is an enzyme, the enzyme's substrate can also be provided in
a separate package of a system. A solid support and one or more
buffers can also be included as separately packaged elements in
this system.
EXAMPLES
The following examples are given for illustrative purposes only and
do not in any way limit the scope of the invention.
1. Purification of Single-Stranded Nucleic Acid Binding Protein
Single-stranded nucleic acid binding protein (Eco SSB) was purified
from E. coli strain K12-H1-TRP/pTL119A-5 essentially as previously
described by Lohman et al., Biochem., 25: 21-25 (1986), according
to the following procedure.
a. Cell Growth and Induction
E. coli strain K12-H1-TRP/pTL119A-5 was grown at 30 degrees
centigrade (30.degree. C.) in media containing 10 grams per liter
(g/l) bactotryptone, 5 g/l yeast extract, 5 g/l NaCl and 2 ml/l of
a solution containing 50 mg/ml thiamine. Single colony isolates of
SSB-coli were admixed with 100 mls of media and grown overnight at
30.degree. C. to produce an overnight culture. 5 ml of the
overnight culture was admixed per 1 liter of media in a 2 liter
flask and the admixture maintained at 30.degree. C. until the
bacterial cells had grown to an optical density at 600 nanometers
(O.D. 600) of 0.4 to 0.6 to produce a log-phase culture. 18 liters
of log-phase culture were produced by this procedure.
The log-phase culture was then heat shocked by maintaining the
culture at 42.degree. C. for thirty minutes. The heat shocked
culture was maintained for four to five hours at 37.degree. C.
Thereafter culture was placed on ice and the bacterial cells
collected from the culture by centrifugation at 5,000 rpm in a J-6B
rotor (Beckman Instruments, Palo Alto, Calif.) for ten minutes at
4.degree. C. All buffers and procedures utilized hereafter were at
4.degree. C. unless otherwise noted. The resulting bacterial cell
pellet was resuspended in a 0.5 liter solution containing 10 mM
Tris-HCl at pH 8.0, 1 mM ethylenediaminetetraacetic acid (EDTA) and
0.1M NaCl. The resuspended pellet was centrifuged for ten minutes
at 5,000 rpm in the same rotor at 4.degree. C., and the resulting
cell pellet was resuspended in 500 ml of a lysis solution
containing 50 mM Tris-HCl at pH 8.3, 0.2M NaCl, 15 mM spermidine
trichloride, 1 mM EDTA and 10% sucrose. The resulting cell pellet
suspension was frozen by addition drop by drop to liquid nitrogen
and stored at -70.degree. C. until used.
b. Cell Lysis
The frozen bacterial cells were thawed overnight to 0.degree. C. in
a freezer. The cells were then thawed in a warm water bath with
gentle mixing at slightly below 10.degree. C. Freshly prepared 0.1M
phenylmethylsulfonyl fluoride (PMSF) was added to a final
concentration of 0.1 mM. A solution containing 10 mg/ml of lysozyme
was added to the solution to a final concentration of 200 ug/ml.
The resulting solution was maintained at 4.degree. C. for thirty
minutes. A freshly prepared solution of 4% sodium deoxycholate in
H.sub.2 O was admixed to the solution to a final concentration of
0.05%. The resulting solution was maintained at 15.degree. C. for
thirty minutes to lyse the bacterial cells and produce a lysate.
This lysate was sonicated for three minutes at 50% duty cycle on a
power setting of 10 using a model 250 sonicator manufactured by
Branson (Danbury, Conn.). The lysate was then centrifuged in a SA
600 rotor (Sorval, Dupont, Wilmington, Del.) at 14,000 rpm for
sixty minutes at 4.degree. C. The resulting supernatant was
collected and termed fraction I.
c. Purification of Cell Lysate
A solution containing 10% Polymin P (PEI; polyethylenimine) at pH
6.9 (Sigma Chemical Corp., St. Louis, Mo.) was slowly admixed with
250 ml of fraction I to a final concentration of 0.4%. The
resulting solution was stirred gently for fifteen minutes to
precipitate protein. The precipitate was collected by
centrifugation in 60 ml capped tubes in a SA 600 rotor at 14,000
rpm for twenty minutes at 4.degree. C. The resulting pellet was
gently resuspended in 250 ml of a solution containing 50 mM
Tris-HCl at pH 8.3, 1 mM EDTA, 0.4M NaCl and 20% glycerol by first
gently breaking the pellets with a tissue homogenizer and then
slowly stirring the solution at 4.degree. C. for thirty minutes.
After the pellet appears to be completely resuspended, the solution
is stirred for an additional fifteen minutes at 4.degree. C. The
insoluble debris is then removed by centrifugation at 14,000 rpm in
a SA 600 rotor for twenty minutes at 4.degree. C. The resulting
supernatant is collected and termed fraction II.
Solid ammonium sulfate was slowly added over a thirty minute period
to fraction II with constant stirring at 4.degree. C. until
approximately 27% saturation with ammonium sulfate was reached.
This solution was stirred for an additional thirty minutes at
4.degree. C. and then centrifuged for thirty minutes in 60 ml
capped tubes at 14,000 rpm in a SA 600 rotor at 4.degree. C. The
resulting pellets were gently resuspended in 225 ml of a solution
containing 50 mM Tris-HCl at pH 8.3, 1 mM EDTA, 0.3M NaCl and 20%
glycerol. After the pellet is completely resuspended the solution
is centrifuged at 14,000 rpm for twenty minutes at 4.degree. C. in
a SA 600 rotor to remove any insoluble material. The resulting
supernatant is termed fraction III.
Fraction III containing approximately 225 mg protein in 225 ml was
dialyzed against 4 liters of dialysis solution containing 50 mM
Tris HCl at pH 8.3, 1 mM EDTA, 150 mM NaCl and 20% glycerol for 12
to 18 hours. A portion of the dialyzed fraction III containing
about 50 mg protein was diluted in the dialysis solution until its
conductivity was brought to within 10% of the conductivity of
dialysis solution, and was then applied to a 5.0.times.10.0 cm P-11
phosphocellulose column (Whatman Inc., Clifton, N.J.) that had been
previously activated according to the manufacturer's instructions
and equilibrated with dialysis solution. The dialyzed and diluted
fraction III was loaded onto the P-11 phosphocellulose column at a
flow rate of 6 ml/minute. The phosphocellulose column was washed
with 400 ml of column buffer containing 50 mM Tris-HCl at ph 8.3, 1
mM EDTA, 150 mM NaCl and 20% glycerol to remove any proteins that
did not bind to the phosphocellulose column. Proteins that bound
the P-11 phosphocellulose column were eluted with a linear gradient
of KCl ranging from a concentration of 0M to 2.0M in column buffer.
Fractions containing 10 ml of eluate were collected and the optical
density at 280 nm measured. Peak fractions were pooled and dialyzed
against a 4 liter solution containing 20 mM Tris-HCl, pH 8.3, 1 mM
EDTA, 500 mM NaCl, 1 mM beta-mercaptoethanol and 50% glycerol and
stored at -20.degree. C. until used. The resulting solution
contained Eco SSB protein at a concentration of 1 mg per ml and was
free of nuclease contamination.
2. Detection of Specific Nucleic Acid Sequences in Genomic DNA
using the Polymerase Chain Reaction
Genomic DNA was purified by phenol extraction and alcohol
precipitation according to the procedures of Ausubel et al, Current
Protocols in Molecular Biology, John Wiley and Sons (1987), from
blood obtained by tail bleed from a transgenic mouse having about 1
to 2 copies of a lambda transgene vector, and is referred to as
lambda transgene mouse genomic DNA. Genomic DNA was also obtained
from human white blood cells by the procedure of Miller et al, Nuc.
Acid Res., 16: 1215 (1988), and referred to as human genomic
DNA.
Polynucleotide primers having the deoxyribonucleotide sequences
shown in Table 1 below were prepared by chemical synthesis using a
model 381A polynucleotide synthesizer (Applied Biosystems, Inc.,
Foster City, Calif.) according to the manufacturer's
instructions.
TABLE 1 ______________________________________ Polynucleotide
Designation Nucleotide Base Sequence
______________________________________ pr 886
5'-TCTGGCTCCAGCCAAAGCCACCCTAG-3' pr 887
5'-GGCTGAGCCCAGTGCCTCCTTGAGTA-3' pr 4266
5'-GGTGGCGACGACTCCTGGAGCCC-3' pr 19012
5'-GACAGTCACTCCGGCCCGTGCGG-3' pr 1A 5'-TGTAAAACGACGGCCAGTGGGGCGGCC
ACAATTTCGCGCCAAACTTGACCG-3' pr 1B 5'-CAGGAAACAGCTATGACCGTGGGCAGC
CTGCGCCCGTTTGGGTCC-3' pr 2A 5'-TGTAAAACGACGGCCAGTCAAGGGATA
ATGTTTCGAACGCTGTTT-3' pr 2B 5'-CAGGAAACAGCTATGACCCCCAGATTA
ACACGGAAAACTTTCCATTTA- 3' pr 737 5'-CGACGACTCGTGGAGCCC-3' pr 738
5'-GACAGTCACTCCGGCCCG-3' ______________________________________
A hybridization reaction admixture was formed by combining the
following reagents in a sterile 0.5 milliliter (ml) microfuge tube:
(a) 69 microliters (ul) of sterile, autoclaved H.sub.2 O; (b) 10 ul
of 10.times. reaction buffer containing 500 mM KCl, 100 mM Tris-HCl
(pH 8.3), 15 mM MgCl.sub.2, and 0.1% sterile gelatin, (c) 8 ul of a
solution containing 2.5 mM each of the deoxynucleotidetriphosphates
(dNTP's) dGTP, dCTP, dTTP and dATP; (d) 2 ul of a solution
containing 250 ng each of two polynucleotide primers, selected from
Table 1 and being indicated in the legends to FIGS. 1-4, (e) 10 ul
of DNA template dilution buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA,
10 mM NaCl) containing genomic DNA as template, the particular DNA
sample and amount being indicated in the legends to FIGS. 1-4; and
(f) zero to 1.0 ul (as indicated) of a solution containing 1
milligram (mg) per ml of Eco SSB protein prepared in Example 1 and
free of nuclease contamination.
The hybridization reaction admixture was heated to 94.degree. C.
and maintained at 94.degree. C. for 3 min to denature the duplex
DNA (genomic DNA) present and form single-stranded templates. Then
the admixture was cooled to 54.degree. C. and maintained at
54.degree. C. for 3 min to allow hybridization to occur and form
duplex DNA. The hybridized admixture was then centrifuged for 10
seconds, in a microfuge at about 12,000.times. g to collect
condensation off the microfuge tube walls, and 0.5 ul of a solution
containing 2.5 units of either Taq DNA polymerase or AmpliTaq.TM.
DNA polymerase (Perkin-Elmer Cetus, Norwalk, Conn.) was admixed to
form a primer extension reaction admixture.
Each microfuge tube containing the above-prepared primer extension
reaction admixture was overlayed with 50 ul of mineral oil and then
placed into a DNA Thermal Cycler (Perkin-Elmer Cetus, Norwalk,
Conn.) and subjected to the following temperature and time
conditions: (1) 72.degree. C. for 1.5 min to allow for primer
extension, (2) 91.degree. C. for 1 min to heat denature the duplex
DNA and (3) 54.degree. C. for 2 min to allow single-stranded
nucleic acids to hybridize. Thereafter the same tubes were
subjected to further cycles of conditions (1), (2) and (3) for a
total of 30 cycles according to the manufacturer's instructions.
The cycled tubes were then maintained at 72.degree. C. for 10 min,
and then maintained at 4.degree. C. for 12 hours.
The contents of each primer extension reaction admixture was then
analyzed on 6% polyacrylamide gels in 1.times. TBE by loading 35 ul
of admixture sample and 5 ul of 10.times. sample buffer onto an 8
centimeter gel, electrophoresing the gel at 100 v for about 1 hr,
and staining the electrophoresed gel with ethidium bromide to
visualize the electrophoresed nucleic acids.
The sensitivity of a PCR nucleic acid detection system using Eco
SSB was determined using the above-described procedures and with
the following additional details. The hybridization reaction
admixture was prepared containing varying amounts of lambda
transgene mouse genomic DNA, from 0.001 ng to 1000 ng. Carrier DNA
comprising calf thymus DNA (100 ng) was also admixed to each tube
containing less than 100 ng of genomic DNA. Duplicate sets of
hybridization reaction admixtures were prepared with the varying
amounts of genomic DNA; one set contained 250 ng (0.25 ul) of Eco
SSB and the other set did not contain Eco SSB. The polynucleotide
primers used were pr 4266 and pr 19012, whose sequences are shown
in Table 1. Taq DNA polymerase was admixed in each tube and the PCR
reaction mixtures were then subjected to PCR cycles as described
above. The results of the sensitivity analysis are shown in FIGS.
1A and 1B.
By use of primers pr 4266 and pr 19012, an amplified nucleic acid
fragment comprising a duplex DNA molecule that corresponds in
sequence to the target specific nucleic acid is detected having a
size of about 520 base pairs (bp), and is indicated in FIGS. 1A and
1B by the arrow. In the absence of Eco SSB (FIG. 1A), the detected
duplex DNA molecule was not a significant component above the level
of detection of non-specific DNA molecules until at least 100 ng of
template DNA were present in the hybridization and primer extension
reaction admixtures. In the presence of 250 ng of Eco SSB, the
detection of nonspecific DNA molecules was substantially reduced
and the limit of detection (sensitivity) for the target sequence
was at least as low as 0.1 ng of template DNA (lane 3, FIG.
1B).
The amount of Eco SSB to be added to a PCR nucleic acid detection
system for effective improvement of detection was also determined
by the above-described procedures. The hybridization reaction
admixture was prepared using from 10 ng to 10,000 ng of Eco SSB in
each tube, including AmpliTaq.TM. DNA polymerase, 100 ng of
template lambda transgene mouse genomic DNA and the primers pr 4266
and pr 19012, and the PCR cycles were conducted as before. The
results of varying the Eco SSB concentration are shown in FIG.
2.
The results in FIG. 2 show that Eco SSB is effective to improve
PCR-based detection of specific nucleic acids over a wide range of
Eco SSB protein concentrations, from below 10 ng to above 5 ug per
100 ul of hybridization reaction admixture. Particularly desirable
results were obtained in which nonspecific bands were substantially
reduced at concentrations of Eco SSB above 100 ng and below 2 ug
per 100 ul of hybridization reaction admixture.
The requirements for stringency in the hybridization reaction were
evaluated when Eco SSB is used in a PCR-based detection system by
conducting the hybridization reaction at either 42.degree. C. or
54.degree. C. The method was conducted as described above with the
following changes. Hybridization reaction tubes were prepared
containing (1) 100 ng of human genomic DNA and the primers pr 886
and pr 887, (2) 100 ng of lambda transgene mouse genomic DNA and
the primers pr 4266 and pr 19012, or (3) 100 ng of the mouse DNA
and the primers pr 737 and pr 738 (18-mers), and further containing
either 250 ng of Eco SSB (+) or no Eco SSB (-). Duplicate sets of
the above tubes were prepared, each containing Taq DNA polymerase,
and were subjected to the above PCR cycles in which the
hybridization (annealing) temperature was either 42.degree. C. or
54.degree. C. The results of the hybridization stringency
determination are shown in FIG. 3.
Using the mouse genomic DNA in combination with either the primer
set pr 4266 and pr 19012 (each 23-mers) (lanes 5, 6, 11 and 12) or
the primer set pr 737 and pr 738 (each 18-mers) (lanes 3, 4, 9 and
10) in the PCR system with Eco SSB, a specific duplex DNA of about
520 bp is detected (FIG. 3, Lanes 4, 6, 10 and 12). Using the human
genomic DNA in combination with the primer set pr 886 and pr 887 in
the PCR system with Eco SSB, a specific duplex DNA of about 1800 bp
is detected (lanes 7 and 8). However, in the absence of Eco SSB,
the specific sequences are either not detected (lanes 1, 3, 7 and
9) or is detected in a manner in which there are significantly more
nonspecific bands when compared to detection with Eco SSB (lanes 5
and 11 compared to lanes 6 and 12).
The effect of temperature on stringency of hybridization is well
characterized. At lower temperatures, higher amounts of mismatched
base pairs are tolerated in a hybridized duplex. The effect of
lower stringency therefore is to increase the occurrence of duplex
formation between single-stranded nucleic acid molecules that do
not have homology or even substantial complementarity. That is,
under conditions of low stringency a duplex can be formed between a
primer/probe and a nucleic acid sequence that is not the target
sequence desired, ie, a nonspecific hybridization product.
Nonspecifically hybridized duplex DNA molecules can initiate primer
extension and lead to the formation of nonspecific amplified
nucleic acid molecules.
The effects of a hybridization temperature that produces conditions
of lower hybridization stringency on a PCR detection system
containing Eco SSB is shown in FIG. 3. As before at 54.degree. C.,
Eco SSB substantially improved the signal-to-noise ratio of
detection of specific nucleic acids over non-specific nucleic acids
when PCR was conducted using a 42.degree. C. hybridization
temperature. Whereas reducing the hybridization temperature lowered
the stringency and therefore increase the nonspecific signal
(compare lanes 9 to lane 3), Eco SSB substantially reduced the
amount of detectable nonspecific duplex DNA under conditions of low
hybridization stringency such as 42.degree. C. (compare lane 3 to
4, or lane 5 to 6). Therefore the use of Eco SSB in a PCR detection
system or method allows for less precision in determining the
critical hybridization temperature and hybridization stringency
required for conducting PCR or other hybridization based
methods.
The results in FIG. 3 also indicate that primers having shorter
nucleotide lengths produce greater levels of nonspecific
hybridization. This is not unexpected because primer length will
effect the hybridization temperature (Tm). For example, compare
lane 11 to lane 9, or compare lane 5 to lane 3, in which a 23
nucleotide primer exhibits more specificity than an 18 nucleotide
primer. When using Eco SSB, the 18 nucleotide primer detects a
specific sequence (lanes 3 and 10) not detectable in the absence of
Eco SSB (lanes 2 and 9), at both 54.degree. C. and 42.degree. C.
Taken together, these data show that the use of Eco SSB improves
specificity and reduces the need for precision in choosing and
controlling hybridization stringency in a PCR detection method,
whether the determinant of stringency is hybridization temperature
or amount of polynucleotide complementarity.
Additional studies were conducted on primers having regions of
non-homology to the target sequence to determine the effect of Eco
SSB on detection of the target sequence in a PCR detection method
using primers having substantial complementarity. Hybridization
reaction admixtures were prepared by the above-described methods
and using Taq DNA polymerase, except that 100 ng of human genomic
DNA was admixed with the primer pair pr 1A and pr 1B, or with the
primer pair pr 2A and pr 2B, in the presence (lanes 2 and 4) or
absence (lanes 1 and 3) of 250 ng of Eco SSB, and the prepared
admixtures were subjected to the above PCR cycles with a 54.degree.
C. hybridization temperature. The results are shown in FIG. 4.
The two primer pairs have sequences that were derived from the
human dihydrofolate reductase gene (DHFR), at exon 1 (pr 1A and pr
1B), or at exon 2 (pr 2A and 2B). However, each primer has 18
nucleotide residues at their 5' ends that are not homologous to the
DHFR gene, but rather correspond in sequence to portions of a
universal lambda primer. At the 3' end of each primer there is a
stretch of 33 nucleotides (pr 1A), 27 nucleotides (pr 1B), 27
nucleotides (pr 2A), or 30 nucleotides (pr 2B) that correspond to
portions of the human DHFR gene. Thus, when specifically hybridized
to the human genomic DNA DHFR gene, the duplex containing these
primers has a 5' "tail" region comprising 18 nucleotides that is
not complementary or hybridized to the template and is attached to
the remaining 3' portion comprising 27 to 33 nucleotides that is
hybridized to the DHFR gene. The above DHFR primers are therefore
examples of primers having substantial complementarity with the
target sequence.
As shown in FIG. 4, the two DHFR primer pairs pr 1A and pr 1B (lane
3) or pr 2A and pr 2B (lanes 1 and 2) detect a specific nucleic
acid sequence by the PCR method that produces an amplified duplex
DNA molecule of about 250 bp. Although the pr 2A and pr 2B primer
pair yielded a specific band both in the presence or absence of Eco
SSB (lanes 1 and 2), the pr 1A and pr 1B primer pair only yielded a
specific band when Eco SSB was present (lane 3). Although the
reason for this difference between the two primer pairs is not
presently identified, an improvement in the PCR detection method
was demonstrated by the data in FIG. 4 for one of the two primer
pairs tested by including Eco SSB in the PCR method. These results
therefore demonstrate that primers with substantial but not
complete complementarity are effective for use in combination with
an SSB protein in the presently disclosed PCR detection method.
The foregoing description and the examples illustrate the present
invention but are not intended to limit the scope of the invention.
Those skilled in the art recognize modifications and variations of
the exemplified embodiments that are within the spirit and scope of
the invention described and claimed herein.
* * * * *